Tumor Biology Laboratory
Transmission Electron Microscopy
Standard Operating Procedure for cell culture sample preparation for
Transmission Electron Microscopy Analysis
Materials
Glutaraldehyde
sodium cacodylate
agar
Osmiun tetroxide
aqueous uranyl acetate
resin Epon Araldite
Propylene oxide
ethanol
Pellet cells in suspension
1. Gently pellet cells (800 g for 5-10 minutes)
2. Remove most of the culture medium and fix the cells adding excess volume (5-10
times the cell volume) of fixative and resuspend the cells in the fixative
3. Primary fixation:
a. 2.5% gluteraldehyde in 0.1 M sodium cacodylate at ph 7.0 from started at
room temperature and continued at 40 C for 30 min
PPE: Use Lab coat, nitrile gloves, mask
Engineering Measures: The technique will be conducted in the hood to
ensure good ventilation when working with toxic chemicals.
4. Cells should be pelleted in eppendorf tubes (3-5 min at 5,000 rpm)
5. Decant fixative.
6. Rinse:
a. Wash in 0.1 M buffer that has been adjusted to the osmolarity of sample
for 5 min.
7. Resuspend sample and leave for 10 min.
8. Prepare a 1 % solution of high strength agar in distilled water by bringing to boil
and stirring
9. Decant buffer from sample tubes and take them and the agar solution to the
centrifuge
10. When agar solution has cooled to ~ 600 C quickly and fill each tube with it.
11. Resuspend samples and spin them at full speed for 30 secs-1 min (max 4 samples
at a time of agar will set before the sample can spun to the bottom to the tube)
12. Cool the tube on a beaker of cold water
13. Remove agar plug with a mounted needle (or wood sticks) and cut off the end
containing the sample
14. Cut up the sample in agar (1 mm cubes) as for embedding to improve penetration
of solvents and place in 0.1 M sodium cacodylate buffer
Tumor Biology Laboratory
Transmission Electron Microscopy
Post fixation:
15. 1% Osmium tetroxide in 100mN phosphate buffer (or cacodylate buffer 0.1 M) 30
min
16. Wash:
a. Wash at least 5 times in distilled water (remove phosphate ions to prevent
Uranyl precipitation)
17. Post fixation:
a. Stain with 2% aqueous uranyl acetate for 30 min at 40 C in dark
18. Prepare resin Epon Araldite:
a. Medcast 12.5 mL (15.11 g ) X 2 (30.22 g )
b. Araldite 7.5 mL (23.59 g ) X 2 (47.18 g )
c. DDSA 27.5 mL (51.12 g ) X 2 (102. 24 g )
d. DMP-30 1.0 mL (52.19 g ) X 2 (104.38 g)*
Note : Do not add the accelerator (DMP-30) until the end, not needed for infiltration just
for the embedding
19. Dehydration:
a. Series of ethanols and propylene oxide
30 % ethanol 5 min
50 % ethanol 5 min
70 % ethanol 5 min
90 % ethanol 5 min
100 % ethanol 10 min (3 times)
11. Resin embedding:
Propylene oxide 2 times each 10 minutes (do not expose to air, or plastics)
Resin infiltration
`
2:1 mix propylene oxide : resin 30min
1:1 mix propylene oxide : resin 30min
1:2 mix propylene oxide : resin 30min
100% resin
overnight
Change in to fresh resin
30 min
Embedding Fresh resin in em bedding moulds – remember to label samples
Polymerize 48 hrs at 52 0 C
Cut with ultramicrotome and stain with uranyl and lead citrate
Waste Disposal: All regulated chemicals must be disposed of in a container and
stored in SAA for the TSO pickup. All non-regulated waste can be disposed of in the
cold trash.
Housekeeping:
The work bench must periodically be wiped down to maintain good housekeeping.
Always use a wet technique for wiping up residual material and place cleaning
materials in a zip lock bag prior to disposal.
Tumor Biology Laboratory
Transmission Electron Microscopy
Fixing Cells with Fresh Galdehyde
Gluteraldehyde Hazzards: Carcinogenic at low doses. Fumes and contact are
dangerous.
Protection: wear gloves when handling gluteraldehyde. Wear mask and goggles when
preparing from powder. Mix in chemical waste hood.
Waste: Discard gluteraldehyde by emptying into hazardous waste container. Store in
chemical waste hood until picked up by waste disposal.
Spill clean-up: refer to MSDS. Any spill over 25 μl is considered hazardous.
Storage: Store powder on chemical shelf. Store liquids in opaque containers at 4ºC. Use
within five days. Label all containers as carcinogenic.